Anti-proliferative and immunomodulatory potencies of cinnamon oil on Ehrlich ascites carcinoma bearing mice

Cinnamon is a well-known natural spice and flavoring substance used worldwide. The objective of the present work is to explore the possible antitumor and immunomodulatory potencies of cinnamon essential oil (Cinn) on Ehrlich ascites carcinoma (EAC). A total of fifty female Swiss albino mice were sub-grouped into five groups (n = 10), namely, normal (a non-tumorized and non-treated) group; EAC-tumorized and non-treated group; Cinn (non-tumorized mice received Cinn, 50 mg/kg per body weight daily) group; a group of EAC-tumorized mice treated with Cinn and the final positive control group of EAC-tumorized mice received cisplatin. Eight compounds were identified from Cinn using UPLC-MS-Qtof and NMR analysis. Compared to EAC untreated group, Cinn successfully (P < 0.05) inhibited tumor growth by reducing tumor cell count (45%), viability (53%) and, proliferation accompanied by the inhibition of tumor growth rate. Moreover, a significant (P < 0.05) arrest in the cell cycle at G0/G1 phase was noticed following Cinn treatments (~ 24.5%) compared to EAC group. Moreover, Cinn markedly evoked an antitumor immune response by elevating the percentage of splenic T helper (CD3+CD4+) and T cytotoxic (CD3+CD8+) cells. It is noteworthy that Cinn treatments significantly restored different hematological alterations as well as liver and kidney functions in EAC-tumorized mice. In conclusion, results suggest that Cinn has a good antitumor and immunostimulatory potencies against Ehrlich ascites carcinoma in vivo. The mechanism underlying its antitumor activity may be attributed to its immunostimulatory effects which increase its potential as a promising anticancer candidate.


Results
Structure determination of secondary metabolites. Eight compounds were identified from cinnamon oil as shown in Table 1 and Fig. 1, 2.

LC-MS-MS and NMR analysis. Cinnamaldehyde, elucidated from Cinnamon oil displayed an [M + ] ion
at m/z 133.0787 in the ( +) HRESIMS spectrum consistent with the molecular formula C 9 H 8 O. 1 HNMR data ( Table 2, Figs. 3, 4) exhibited one deshielded aldehydic signal at δ H 9.74 ppm, two aromatic peaks at δ H 7.43 and 7.57 ppm correlated to five protons of the benzene ring. Two protons at δ H 7.48 ppm doublet with J coupling 16 Hz and doublet-doublet J coupling 16.0, 7.7 Hz indicating the alpha-beta unsaturated ketone. 13 CNMR quaternary carbon at δ C 128.41 was correlated to aromatic ring carbon, two deshielded carbons at δ C 193.59 and 152.62 ppm related to the aldehydic carbon and beta carbon of alpha-beta unsaturated ketone and alpha carbon showed at δ C 128.53. Finally, the aromatic five carbons were displayed at δ C 129.02 and 128.4.

Effect of cinnamon oil on tumor burden and the survival of EAC-bearing mice. The results
revealed that treatment with Cinn led to a significant reduction (65%) in peritoneal ascites volume compared to the EAC group (Fig. 5A, B). The tumor cell counts (Fig. 5C) and cell viability (Fig. 5D) were reduced compared to the untreated group by approximately 45 and 53% respectively (Fig. 5A). Moreover, the treatment with Cinn induced about 53.5% tumor growth inhibition when compared with the EAC-bearing mice (Table 3). However, mean survival time (MST) of EAC-bearing mice treated with Cinn was 25 days while the EAC group showed 22 days. The increase in life span (ILS) was recognized to about 13.64% in Cinn-treated group in respect to the EAC group (Table 3, Fig. 5E). www.nature.com/scientificreports/     www.nature.com/scientificreports/ Physical parameters. Results revealed that treatment of non-tumorized mice with cinnamon oil led to a significant (P < 0.05) decrease in body weight and spleen index and a significant (P < 0.05) increase in thymus index compared to the control group. Cisplatin treatment of EAC group decreased (P < 0.05) the body weight accompanied by a significant increase in thymus index, while caused a non-significant change in spleen index relative to EAC control group. Treatment of EAC-bearing mice with cinnamon oil led to a decrease in body weight (P < 0.05) accompanied by significant increase in spleen index, while causing non-significant changes in thymus index compared to EAC control group and Cis group (Fig. 6). Finally, Cinnamon oil attained to restore animals' body weight and spleen index to be near to those of normal non-tumorized and non-treated mice.
Quantification of EAC apoptosis/necrosis. In order to assess the antitumor effect of Cinn on EAC cells, flow cytometric analysis of apoptosis was performed (Fig. 7A). The results clarified that Cinn significantly (P < 0.05) reduced the apoptotic events by ~ 45.5 and 21% compared to EAC-bearing mice and Cis-treated group respectively. However, the necrosis was significantly increased (~ 434.5 and 520%) in the Cinn-treated group related to the EAC-bearing mice and Cis-treated group respectively (Fig. 7C).

Cell cycle distribution of EAC.
Considering the effect of Cinn on EAC cells using the flow cytometric analysis of the cell cycle distribution in treated and control groups (Fig. 7B), Cinn showed a significant (P < 0.05) arrest of the cell cycle in the G 0 /G 1 phase supported by an elevated accumulation of cells' population (~ 24.5%) compared to EAC group. However, Cis treatment caused a significant (P < 0.05) S phase arrest as confirmed by the increased EAC population by ~ 197% compared to the EAC group (Fig. 7D).

Assessment of EAC cells' DNA fragmentation.
The extracted total genomic DNA samples of control and treated EAC groups were resolved on agarose gel (Fig. 8A). The control group did not show DNA fragmentation. However, the tumor cells from Cinn-treated mice showed evidence of necrosis which was indicated by the presence of smeared DNA fragmentation. Furthermore, the Cis-treated group revealed a significant reduction in cells reflected by the decreased optical density of isolated DNA. This reduction reached ~ 80 & 73.5% in the Cinn-treated group compared with EAC-bearing mice and Cis-treated group respectively (Fig. 8B).

Antiproliferative effect of cinn on EAC cells.
To investigate the antiproliferative potency of Cinn on EAC, Ki67-nuclear protein expressed cells were evaluated by the flow cytometric analysis in control and treated groups (Fig. 9). Results revealed that the control EAC group exhibited the highest proliferative properties with 49.8 ± 1.8% of the Ki67 positive cells. However, Cinn-treated mice displayed a significant decline in the tumor cell proliferation rate with Ki67 expression percentage reaching 34.4 ± 2.2% with respect to EAC-bearing ani-   (Fig. 10, 11). On the other hand, treatment of tumorized mice with Cinn decreased (P < 0.05) the percentage of T regulatory (CD4 + CD25 + ) splenocytes by approximately 12.8% compared to 48.4% of the control group (Fig. 12). While, cinnamon oil treatment of EAC-bearing mice caused non-significant changes in the percentage of  www.nature.com/scientificreports/ CD3 -CD8 + CD56 + spleen cells compared to control mice (Fig. 13). In order to link the Cinn treatment with the immune cell-mediated anticancer effect, the correlation between immune cell changes and Cinn anti-cancer effects was evaluated. Ki67 expression was significantly correlated with the percentage of CD3 + CD4 + (R = 1.00; P-value = 0.005). However, the antiproliferative potency was supported by a negative correlation of Ki67 expression with the percentage of CD3 + CD8 + cells (R = − 0.932; P-value = 0.236). Furthermore, there was a negative correlation between the percentage of CD3 -CD8 + CD56 + and both of apoptotic and necrotic events (R = -0.924; P-value = 0.250, R = − 0.833; P-value = 0.374, respectively).

Hematological parameters.
The hematological changes were observed in treated EAC-bearing mice and control ( Table 4). The results proved that the mice received Cinn exhibited a significant (P < 0.05) elevation in the count of total white blood cells (WBCs), hematocrit value and relative granulocyte count. However, the decreased (P < 0.05) relative lymphocyte count was noticed, while, there were no significant changes in the count of red blood cells (RBCs), hemoglobin content (Hb) and platelet count compared to normal healthy control. Moreover, cisplatin treatment caused a significant decrease in the count of total white blood cells (WBCs) and relative granulocyte count accompanied by an increase (P < 0.05) in platelet count and relative lymphocyte count. Non-significant changes in the count of red blood cells (RBCs) and hemoglobin content compared to the EAC control group. Furthermore, treatment of EAC-bearing mice with cinnamon oil led to a significant (P < 0.05) improvement in WBCs count, RBCs count, hemoglobin content, platelet count, relative lymphocytes and granulocytes when compared to the EAC control group and Cis-treated group. Collectively, treatment with cinnamon oil partially succeeded to restore many hematological parameters toward the normal values.
Biochemical evaluations. Results showed in Table 5 revealed a non-significant changes in ALT, AST, urea and creatinine serum levels in Cinn-treated mice when compared to normal healthy control. Tumor growth was correlated with changes in liver and kidney functions as approved by the elevated (P < 0.05) levels of serum AST, ALT, urea and creatinine. Cisplatin treatment significantly deminished serum ALT and AST concentrations, while caused a non-significant changes in urea and creatinine serum levels compared to EAC group. Cinn treatment of EAC-bearing mice led to an improvement (P < 0.05) in ALT, AST and urea serum levels accompanied by non-significant change in serum creatinine levels when compared to EAC group and Cis-treated group. Taken together, cinnamon oil attained to ameliorate liver and kidney functions toward the normal levels.

Discussion
The conventional chemotherapies associated with serious side effects, which limit their therapeutic application in diverse cancers. Complementary and alternative medicines such as traditional herbal medicine become the corner stone of new medication strategies, especially for cancer cure 23,24 . The advantage of therapeutics from natural origins is that it targets various similar signaling inputs in different physiological events as in the organism of origin and thus playing the same defence mechanism that enables the natural pure elemts to eliminate the harmful insults, and cleare them from the body, and even at larger quantities may have less detrimental toxicity in comparison to synthetic agents 25 . The current study showed that cinnamon oil has anti-tumor effect through reducing tumor cell count, tumor cell viability, ascites volume and the rate of tumor progression. These results were supported by the previous report demonstrating the suppression of melanoma cell line (B16F10 and Clone M3 mouse melanoma cells) in vivo and in vitro by cinnamon 12 . In line, essential cinnamon oil has been proved to exert significant anti-cancer potential towards head and neck squamous cell carcinoma by decreasing tumor weight and increasing tumor inhibition rate in nude mouse model utilizing Hep-2 cells. Moreover, essential Figure 6. Changes in body weight, spleen and thymus indices following treatment with cinnamon essential oil (Cinn, 50 mg/kg b.wt/day, orally) revealing that Cinn essential oil partially succeeded to retrieve body weight, spleen and thymus indices to their normal values. All values were presented as Mean ± SD, (n = 3). Significant differences (P < 0.05) between groups were shown by different letters. Cis, cisplatin (2 mg/kg b.wt, i.p). www.nature.com/scientificreports/ cinnamon oil has been proved to decrease laryngeal carcinoma cell line (Hep-2) tumor burden by 43.5% through suppressing the activity of epidermal growth factor receptor tyrosine kinase 13 . The immune system's major organs are thymus and spleen. The thymus is the main place for T-cell differentiation, maturation, and also linked to humoral immunity. The spleen involved in the production of immune cells and monocytes. Thus, the body's immune function could be reflected by thymus and spleen indices. Herein, the treatment of EAC-bearing animals with cinnamon oil significantly increased the spleen index, and slightly increased the thymus index relative to . The statistical data of treated and control groups of apoptosis/necrosis and cell cycle phases respectively. All values were presented as mean ± SD, (n = 3). Significant differences (P < 0.05) between groups were shown by different letters. Cis, cisplatin (2 mg/kg b.wt, i.p). Similarly, previous studies were in agreement regarding the increased indices of immune organs in response to natural components when compared to tumorized animal models and those received chemotherapeutics 26 . Deregulated proliferation and apoptosis are the key players of all cancer development and they present critical targets for therapeutic interventions 27 . The current results clarified that cinnamon oil caused a significant reduction in tumor cells proliferation through reducing the nuclear protein Ki67 expression. In addition, Cinn led to a significant arrest of cell cycle in G 0 /G 1 phase. This came in accordance with Schoene et al. 28 results exclaiming that cinnamon decreased the proliferation and arrested the cell cycle of myeloid cell lines (Jurkat, Wurzburg, and U937 cells) in G 2 /M phase by decreasing the activity of intracellular phosphatase. Similarly, the in vitro anticancer effect against HNSCC cells and the suppression of the tumor growth in Hep-2 xenograft were documented 19 .
Cinnamaldehyde, an active ingredient of the cinnamon park, was found to arrest Jurkat and U397 leukemia cells in G 2 /M phase 29 and HCT116 colon cancer cells 30,31 . The anti-proliferative potency against MCF-7 cells 30 and both HL60 promyelocytic leukemia cells were investigated too 32 . Furthermore, in vitro G 1 cell cycle arrest was noticed in human metastatic melanoma cell lines (A375, G361, LOX) with cinnamaldehyde administration 33 . Another constituent of cinnamon, cinnamic acid, displayed antioxidant immunomodulatory, anti-inflammatory, and anticancer potencies 34 . The antiproliferative and induction of G 0 /G 1 arrest in lung cancer stem cells has been reported with cinnamic acid treatment 35 . Furthermore, some derivatives of cinnamic acid have been proved to have antitumor and anti-proliferative potencies in vitro against many human cancer cell lines including HT-29, A-549, OAW-42, MDA-MB-23, HeLa 36,37 . Interestingly, Liu et al. 20 investigated possible mechanisms of cinnamaldehyde action in breast cancer treatment; they fundamentally explained the neuroactive ligand-receptor interaction, and NFқ-B, cAMP, PI3K-AKt, PPAR, BDNF signaling pathways. Moreover, cinnamophillin (another constituent of cinnamon oil) was previously reported as a free radical scavenger [38][39][40] which may have contributed positively to the improvement of the health status of treated mice. The current results suggest that the anticancer activity of Cinn may be linked with the inhibition of epidermal growth factor receptor-tyrosine kinase, in line with the earlier literature 19 . Furthermore, it may be attributed to the effects of cinnamaldehyde on reactive oxygen species (ROS) generation 33 or by inhibiting thioredoxin reductase enzymatic activity 30 . The antioxidant properties of the oil constituents 34 may be involved in the antitumor activities 41,42 .
Immune surveillance, the main defense against cancer, allows the immune cells to detect and exclude tumor cells 43 . Helper T cells (CD3 + CD4 + ), cytotoxic T cells (CD3 + CD8 + ), and NK cells were extremely involved in antigen-specific tumor removal [44][45][46] . In this study, results showed a marked elevation in splenic T regulatory cells (CD4 + CD25 + ) accompanied by the significant depletion of cytotoxic T cells and NK cells in EAC-bearing mice relative to normal mice. Cinn treatment of tumorized mice succeeded in partial restoration of the normal percentage of CD3 + CD4 + , CD3 + CD8 + and CD4 + CD25 + splenic cell populations, while causing non-significant changes in CD3 -CD8 + CD56 + splenocytes. Similar to earlier findings, an escape strategy of the tumour cells, the tumour microenvironment was characterised by the proliferation of particular cell types such as regulatory T cells (CD4 + CD25 + ) in order to suppress the protective antitumor immunity [47][48][49][50][51][52][53] . In accordance, Ibrahim et al. 54 www.nature.com/scientificreports/ clarified that tumor development was linked to a decrease in the percentage of splenic CD3 + CD8 + cell population in Ehrlich solid tumor bearing mice. Furthermore, cinnamon effectively restored the balance of T-cell subsets by promoting the proliferation of T helper-1 and T cytotoxic 1 while reducing the proliferation of regulatory T cells in SLTBI mice and augmenting the IFN-γ production and strengthening the antitumor effect against lung metastasis of melanoma in SLTBI mice 55 . The major effector of cytotoxic T lymphocytes and NK cells in attacking the cancer cells is the granzyme B. Granzyme B has several protein targets in cancer cells, and after crossing the endosome membrane, the enzyme is transported to numerous subcellular components, such as the mitochondria, nucleus, and cytoplasm 56 . Among these substrates, the nuclear protein Ki67 is cleaved by granzyme B at multiple sites 57 . This may interprete the antiproliferative potency of cinnamon oil by elevating the percentage of activated cytotoxic T lymphocytes which in turn pump more granzyme B and other enzymes into cancer cells leading to the antiproliferative effect on cancer cells.  www.nature.com/scientificreports/ The liver is a key organ that is extensivly involved in various metabolic and detoxifying functions. Hence, measuring serum levels of the liver enzymes ALT and AST is a useful tool, particularly in the follow-up and monitoring of liver status 58,59 . The results illustrated that Cinn treatment induced a partial improvement in the serum levels of ALT, AST, urea while making non-significant changes in serum creatinine level with respect to EAC-bearing mice and Cis-treated group. In the same line, cinnamon was reported to have ameliorative effect against kidney disorder induced by cypermethrin in male Wistar albino rats, this effect may be owed to cinnamon's antioxidant activity 60 . The ameliorative potency of cinnamon oil against liver toxicity in albino rats with HCC was reported by an obvious decline in serum levels of ALT and AST 61 and in Wistar rats with acetaminophen-induced acute liver toxicity 62 . Cinnamon was found to ameliorate ALT and AST serum levels toward their normal values in paracetamol induced hepatic toxicity in rats 63 , and in CCl 4 entoxicated rats 64 . Moreover, cinnamon succeeded to ameliorate the deteriorations in ALT, AST, urea and creatinine serum levels caused by declofenac sodium and oxytetracycline in male albino rats 65 . Several studies discussed the nephroprotective effect of cinnamon represented in modulating urea and creatinine serum levels 9,[66][67][68] .
In order to estimate the general state of health and the effects of the therapy on the host, hematological changes were evaluated during cancer therapies administration 69 . In this study, tumorized mice treated with Cinn exhibited a marked improvement in the total leucocytic count, RBCs count, hemoglobin content, platelet count, relative lymphocytes and granulocytes when compared to EAC control group and Cis-treated group. These outcomes were in consistent with previous work which reported that cinnamon extract improved the WBCs count, RBCs count, hemoglobin content in Alloxan-induced diabetic female albino rats 70 . Additionally, the improved effect of cinnamon on the hematological parameters in vivo was previously reported 71,72 . Based on current findings, further studies are needed to investigate the effect of cinnamon oil on EAC for different administration doses. Moreover, the oil constituents should be evaluated individually for their anticancer and immunomodulatory potencies. Animals. Female Swiss CD1 mice weighing 24 ± 4 g (6-8 weeks old) were supplied by the National Research Center, Giza, Egypt. All animals were kept in a standard laboratory settings with a 12-h light/dark cycle. The rodent food (El-Haramain rodent diet, Egypt) and water were supplied. Acclimatization to laboratory conditions for 12 days prior to the experiments was done to all mice.

Cell line and experimental design. Ehrlich ascites carcinoma (EAC) cells were supplied by the National
Cancer Institute, Cairo University, Egypt. Serial intraperitoneal (i.p.) inoculation of viable tumor cells (0.5 × l0 6 ) in a saline solution (0.2 mL) was used for cells' maintainence into female Swiss CD1 mice. The viabile cells were detected by the trypan blue (0.4%) assay and counted using a hemocytometer. A total of 50 apparently healthy Swiss female mice were involved in this study, 30 of them were injected i.p. by about 0.25 × l0 6 viable EAC cells 75 . Animals were divided into five groups (n = 10) as the following: Group I, normal mice receiving (i.p.) 0.9% saline solution (0.2 mL); Group II, EAC-bearing mice without any treatments and received corn oil as a vehicle; Group III, normal mice receiving an oral dose of Cinn (50 mg/kg b.wt, daily) 19 ; Group IV, EAC-bearing mice receiving an oral dose of Cinn (50 mg/kg b.wt, daily); Group V, EAC-bearing mice were injected with Cis (2 mg/kg b.wt, i.p) every 48 h according to Salem et al. 76 . Group III was considered as a vehicle for cinnamon oil to investigate the biosafety of the studied dose. Three mice per group were enrolled in the experiments, while the remaining seven mice were used for the mean survival time (MST) and the increase percent in life span (%ILS) investigation. Exeperiments were done in triplicates.

Sampling and preparation of cells.
On the 12 th day, treated mice and controls were anesthetized using halothane inhalation to collect blood from the orbital sinuses, blood samples were collected. Samples were divided into two aliquots, one was allowed to be clotted while the other was kept with EDTA. Sera were obtained after centrifugation at 4000 rpm for 20 min and stored at − 80 °C until use. Mice were then sacrificed by cervical dislocation and the centrifugation at 1000 rpm for 10 min was used for EAC cells harveste. In addition, the spleen was collected and the splenocyte single-cell suspension was prepared after RBCs lysing using ACK lysis buffer according to 77 . The count and viability of EACs and splenocytes were determined using Trypan blue dye exclusion method. Spleen and thymus indices were assessed using the following formula: Organ index = organ weight (g)/ body weight (g) 78 .   Table 4. The effect of Cinn oil treatment on the hematological changes in EAC-bearing mice. Data were showen as mean ± SD (n = 3). Significant differences (P < 0.05) between groups were shown by different letters. EAC, Ehrlich ascites carcinoma; Cis, Cisplatin (2 mg/kg b.wt, i.p); Cinn, Cinnamon essential oil (50 mg/ kg b.wt/day, orally); RBCs, red blood corpuscles; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; WBCs, white blood cells. www.nature.com/scientificreports/ MST of each group was observed by counting the mortality. Specification of the end point of this experiment was the unprompted death of mice. According to the following formula, MST was calculated: MST = (day of first death + day of last death)/2. The percentage of ILS was calculated using the following equation (ILS% = (T − C)/C × 100) 79 .
Where T refers to the MST of treated animals and C represents MST of the control group.
Heamatological and biochemical investigations. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea and creatinine were determined in control and treated groups using the available kits (Human, Max-Plank, Wiesbaden, Germany) and according to the manufacturer instructions. Hematological parameters such as red blood cells (RBCs) count, hemoglobin concentration (Hb), haematocrit value, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBCs) count, platelets count were performed manually on the anticoagulated blood samples 80 .

Total genomic DNA fragmentation in EAC cells. DNA extraction and detection of fragmentation in
control and treated EAC cells were performed using the method of salting-out extraction 81 after some modifications as adjusted by El-Garawani and El-Nabi 82 . About 40 µL of EAC cell suspension from treated and control mice were lysed in DNA lysing buffer at 45 °C for 24 h. Then, proteins and other cellular components were removed by a solution of NaCl (4 M). Nucleic acids precipitation was done using cold isopropanol. The dissolved nucleic acids in Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) were incubated with a loading buffer supplemented with RNase for 30 min. The samples were subjected to ethidium bromide direct staining and resolved on 1.8% agarose gel. The intensity of DNA bands were analyzed using Image J software (Maryland, USA).
Annexin V/Propidium Iodide (PI) labeling for apoptosis quantification. Using a flow cytometry, apoptosis and necrosis were determined in EAC cells according to the Annexin V-FITC Kit (BD Pharmingen, San Diego, CA, USA) instruction manual. Briefly, EACs were collected and washed using PBS, then FITCconjugated Annexin V and PI (Invitrogen, Carlsbad, CA, USA) labeling was done. Sample analysis using a BD Accuri C6, San Jose, CA, USA flow cytometer and its compatible software was performed.
Cell cycle analysis using flow cytometer. The effect of cinnamon oil on the phases of EAC cell cycle was investigated using a flow cytometer. Briefly, tumor cells were washed using ice-cold PBS, ethanol fixed and incubated in PBS containing 1 mg/mL of propidium iodide (PI) and 200 µg/mL of RNase A for 15 min. The cell percentages in sub-G 1 , G 0 /G 1 , S, or G 2 /M phases were assessed using the MODFIT DNA analysis program (Verity Software House, Topsham, ME, USA, version: 2.0). A BD Accuri C6 flow cytometer and its compatible software (San Jose, CA, USA) was used for phase distribution analysis.
Proliferation assay. The proliferation nuclear protein, Ki67, was assessed in EAC cells. According to the manufacturer (Santa Cruz Biotechnology, Inc., Texas, USA), the analysis of positive Ki67-expressed cells was performed using a BD Accuri C6 flow cytometer and its compatible software (San Jose, CA, USA).
Splenocytes phenotypic analysis. The percentages of splenic T-helper CD3 + CD4 + , cytotoxic T-cells CD3 + CD8 + , regulatory T-cells CD4 + CD25 + and NK cells CD3 -CD8 + CD56 + were investigated using flow cytometric analysis. Briefly, following the incubation on ice with the monoclonal antibodies conjugate (mAbs) for 25 min, cells were post-fixed in the dark with 0.3 mL of 1 × CellFIX (BD, Biosciences, San Jose, USA), kept at 4 °C, and then the expression of targeted surface markers was analyzed using a BD Accuri C6 (San Jose, CA, USA) flow cytometer and its compatible software.
Data Analysis. The experiment was done in triplicates; mean ± SD was used for presenting data (n = 3). For statistical analysis, the IBM SPSS statistics program Version 22 for Windows (Armonk, NY, USA) was used. All variables were tested for normality (Kolmogorov-Smirnov& Shapiro Wilk tests) and homoscedasticity (Levene's test). The analysis of variance by one-way ANOVA test was performed followed by Duncan post hoc multiple Table 5. The effect of Cinn treatment on the biochemical parameters in EAC-bearing mice. Data were shown as mean ± SD, (n = 3). Significant differences (P < 0.05) between groups were shown by different letters. EAC, Ehrlich ascites carcinoma; Cis, Cisplatin (2 mg/kg b.wt, i.p); Cinn, Cinnamon essential oil (50 mg/kg b.wt/day, orally); AST, Aspartate aminotransferase; ALT, Alanine aminotransferase.